Novel amino acid sequences, DNA encoding the amino acid sequences, antibodies directed against such sequences and the different uses thereof

ABSTRACT

The invention is directed to a peptide comprising the amino acid sequence substantially as denoted by SEQ ID NO:1 and biologically functional homologues and derivatives thereof.

[0001] This is a continuation-in-part of PCT/IL99/00595, filed Nov. 4,1999, which claims priority from U.S. Provisional Application No.60/107,213, filed Nov. 5, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates to various peptides, homologous toregions of heat shock protein (HSP), to DNA sequences encoding suchpeptides, to DNA constructs comprising the DNA sequences, to antibodiesdirected against peptides of the invention. The invention also relatesto active vaccines comprising a peptide, a DNA sequence or a DNAconstruct of the invention, and to a passive immunization compositioncomprising at an antibody of the invention.

BACKGROUND

[0003] Throughout this application, various publications are referred toby Arabic numerals in parentheses. These publications are incorporatedherein in their entireties and constitute part of the description.

[0004] Adjuvant Arthritis (AA) is an experimental model of autoimmunearthritis which can be induced in susceptible strains of rats such asinbred Lewis or Wistar strains upon vaccination with heat-killedMycobacterium tuberculosis (MT) in complete Freund's Adjuvant (CFA)[1-3]. The disease cannot be induced in resistant strains of rats (e.g.,Brown-Norway; Fisher [5, 6], and Lewis rats develop resistance tore-induction of the disease after recovery from arthritis.

[0005] The inventors have previously shown that resistance to AA can betransferred to a susceptible strain of rats by intravenous infusion ofimmunoglobulins from the resistant strains, and that resistance isassociated with the presence of antibodies against the 65 kD MT heatshock protein (HSP 65) [4].

[0006] Heat shock proteins are a family of highly conserved proteins.There is ˜50% amino acid identity between the Mycobacterial HSP 65 andthe mammalian HSP 60 [21]. The role of the 65 kD heat shock protein (HSP65) of MT in the pathogenesis of autoimmune arthritis, both inexperimental animals [7, 8] as well as in humans [9-11], has beeninvestigated intensively in the past several years. For example, Barkeret al. [32] describe the suppression of arthritogenic immune responsesin mice given HSP65 and pristane. The antigen used to elicit theresponse was full-length HSP65, and no attempt was made to investigatethe effect of specific sub-domains or peptides deriving from thisprotein.

[0007] AA can be passively transferred by a T-cell clone reactive toresidues 180-188 of the MT HSP 65, and in patients suffering fromrheumatoid arthritis (RA), an association between T-cell responses toHSP 65 and early stages of joint inflammation has been found [7, 12-14].Paradoxically, pre-immunization with the mycobacterial HSP 65 leads toresistance to induction of the disease by MT, and this protective effectis believed to be mediated by T cells specific for HSP 65 [7, 15-16].Likewise, although arthritic rats develop vigorous T cell responses toself-HSP and to peptide 180-188 of the MT HSP, neither of these isarthritogenic when injected to arthritis-susceptible rats [15, 17].These results and other suggest that HSP may contain epitopes that aredisease-related and other epitopes that confer resistance [5, 19, 20].Both the pathogenic immune response as well as the protective effectwere attributed to anti-HSP T-cells. The following Examples illustratethe fine epitope specificity of the anti-HSP antibodies of arthritis-susceptible and resistant rats.

[0008] In addition, the inventors have found that naive Lewis rats lackantibodies to certain epitopes of the mycobacterial HSP 65 which arefound naturally in young BN and old naive Lewis rats, and that areacquired by young Lewis rats after recovery from the disease. Analysisof the primary and tertiary structure of the whole MT HSP 65 kD moleculeindicated that these “protective” epitopes are potential B-cell epitopeswith a non-conserved amino acid sequences that are found on the outersurface of the molecule.

[0009] Pre-immunization of Lewis rats with one of the “protective”epitopes prior to induction of the disease induced antibodies againstthe whole molecule as well as resistance to disease induction. Thispeptide corresponds also to the self-HSP 60 epitope to which antibodieswere found in the arthritis -resistant rats, but not in thearthritis-susceptible naive Lewis rats.

SUMMARY

[0010] The present invention relates to a peptide comprising the aminoacid sequence substantially as denoted by SEQ ID NO: 1 and biologicallyfunctional homologues and derivatives thereof.

[0011] More particularly, the invention relates to a peptide having theamino acid sequence substantially as denoted by SEQ ID NO:2 andbiologically functional homologues and derivatives thereof and to apeptide having the amino acid sequence substantially as denoted by SEQID NO:3 and biologically functional homologues and derivatives thereof.

[0012] In addition, the invention relates to a peptide comprising theamino acid sequence substantially as denoted by SEQ ID NO:4 andbiologically functional homologues and derivatives thereof.

[0013] The peptides of the invention can be synthetic peptides andchemically modified peptides.

[0014] The peptides of the invention are capable of conferring immunityagainst autoimmune and/or inflammatory disorders.

[0015] In a further aspect, the invention relates to a nucleic acidsequence encoding a peptide of the invention and to DNA constructscomprising the same.

[0016] In yet a further aspect, the invention relates to vaccinescomprising as active ingredient an effective vaccinating amount of atleast one peptide of the invention, or a nucleic acid according to theinvention. The vaccines of the invention are particularly useful inconferring immunity against autoimmune or inflammatory disorders.

[0017] Still further, the invention relates to antibodies directedagainst the peptides of the invention and to compositions comprisingthem. The compositions of the invention are particularly useful for thepassive vaccination against autoimmune or inflammatory disorders.

[0018] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0019]FIG. 1, Mycobacterium tuberculosis, rat HSP 60 and human HSP 60(sequences P06806, P19227 and P10809, corresponding to SEQ ID NOs:6, 7and 8 respectively), were compared with pileup program fromGCG-Wisconsin Package v9.0. The conserved regions are indicated(consensus). Bold, underlined residues represent the preferred peptides.

[0020]FIG. 2, the GroES heptameric ring is shown in dark gray. The twoGroEL heptameric rings are shown in light gray. Peptides 6-7 (aminoacids 31-52) and 31 (amino acids 181 -197) are also indicated.

[0021]FIGS. 3a-3 b, the location of peptides 6, 7 and 31 in the HSP 65monomer is indicated in a secondary structure configuration (FIG. 3a)and in the space filling mode (FIG. 3b).

[0022]FIG. 4, vaccination against AA with HSP peptides 6, 7 and R5 isshown. PBS was employed as a control.

[0023]FIG. 5, the common motif within peptides 6, 7 and R5, V--E--WG-Pis shown.

[0024]FIG. 6, restriction map of the plasmid pTARGET is shown.

DETAILED DESCRIPTION

[0025] The present invention relates to peptides comprising the aminoacid sequence substantially as denoted by SEQ ID NO:1, and biologicallyfunctional homologues and derivatives thereof.

[0026] Preferably, the peptide according to the first embodiment of theinvention has the amino acid sequence substantially as denoted by SEQ IDNO:2 or the amino acid sequence substantially as denoted by SEQ ID NO:3.

[0027] The invention further relates to a peptide comprising the aminoacid sequence substantially as denoted by SEQ ID NO:4 and biologicallyfunctional homologues and derivatives thereof.

[0028] The invention also relates to a nucleic acid sequence whichencodes a peptide according to the invention.

[0029] More particularly, the invention relates to a DNA sequencecomprising the nucleic acid sequence substantially as denoted by SEQ IDNO:5, and biologically functional derivatives thereof. This nucleic acidsequence encodes a peptide having the sequence substantially as denotedby SEQ ID NO:4.

[0030] The amino acid and nucleic acid sequences are presented inTable 1. TABLE 1 SEQ Amino Acid or ID No Peptide No. Nucleic AcidSequence 1 GPKGRNVVLEKKWGAPTITNDG 2 6 GPKGRNVVLEKKWGAP 3 7VVLEKKWGAPTITNDG 4 R5  TVIIEQSWGSPKVTKDGVTV 5GCCGCCATGGGACCAAAGGGACGCAACGTGG TACTAGAGAAGAAATGGGGCGCGCCGTAGCT CGAGA

[0031] By the term “biologically functional homologues and derivatives”is meant any variations, including deletion, substitution and/orinsertion of an amino acid residue in the amino acid sequences or anucleic acid in the nucleic acid sequences of the invention which wouldnot alter the biological activity of the peptides, or peptides encodedby the nucleic acid sequences, against autoimmune diseases. Thus, thisterm is to be taken to mean peptides with similar structure, peptides ortheir derivatives that are recognized by the protective antibodiesand/or peptides or their derivatives that can induce protectiveantibodies upon immunization.

[0032] The invention further relates to DNA constructs comprising thenucleic acid sequence of the invention or functional homologues andderivatives thereof. The constructs of the invention may furthercomprise additional elements such as promoters, regulatory and controlelements, translation, expression and other signals, operably linked tothe nucleic acid sequence of the invention.

[0033] The invention also relates to a vaccine comprising as activeingredient an effective vaccinating amount of at least one peptide ofthe invention. The vaccines of the invention are particularly intendedto confer immunity against inflammatory and autoimmune diseases, forexample, rheumatoid arthritis or adjuvant arthritis.

[0034] By the term “effective vaccinating amount” is meant an amountsufficient to stimulate the immune system, directly or indirectly, andconfer immunity against inflammatory and autoimmune diseases. Sucheffective amount is determined the severity of the disease, age, sex andweight of the patient, as well as the patient's general condition, andby other considerations known to the attending physician. Preferreddoses, per injection, may be 0.02-2 mg/Kg body weight.

[0035] The vaccines of the present invention may alternatively compriseas the active ingredient at least one nucleic acid sequence according tothe invention.

[0036] The vaccines according to the invention may optionally furthercomprise pharmaceutically acceptable carriers, diluents additives,excipients and adjuvants. By the terms “pharmaceutically acceptablecarriers, diluents additives, excipients and adjuvants” is meant anyinert, non-toxic material that may assist in the efficient delivery ofthe active ingredient.

[0037] The term “antibody” as used in connection with the presentinvention refers to both polyclonal and monoclonal antibodies.Polyclonal antibodies may be generated in rabbits, chicken, mice, rats,sheep, or similar mammals. The generation of polyclonal antibodiesagainst peptides is described in the above-noted Current Protocols inImmunology, Wiley and Sons Inc. Chapter 9.

[0038] Monoclonal antibodies may be prepared from B cells taken from thespleen or lymph nodes of immunized animals, in particular rats or mice,by fusion with immortalized B cells under conditions which favor thegrowth of hybrid cells. For fusion of murine B cells, the cell line Ag-8is preferred.

[0039] The technique of generating monoclonal antibodies is described inmany articles and textbooks, such as the above-noted Chapter 2 ofCurrent Protocols in Immunology. Chapter 9 therein describes theimmunization of laboratory animals with peptides. Spleen or lymph nodecells of these animals may be used in the same way as spleen or lymphnode cells of protein-immunized animals, for the generation ofmonoclonal antibodies as described in chapter 2 therein.

[0040] The term “antibody” is also meant to include both intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab′)2, which are capable of binding antigen. Fab and F(ab′)2 fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding than anintact antibody.

[0041] An antibody is said to be “directed against” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

[0042] An “antigen” is a molecule or a portion of a molecule capable ofbeing bound by an antibody which is additionally capable of inducing ananimal to produce antibody capable of binding to an epitope of thatantigen. An antigen may have one or more than one epitope. The specificreaction referred to above is meant to indicate that the antigen willreact, in a highly selective manner, with its corresponding antibody andnot with the multitude of other antibodies which may be evoked by otherantigens.

[0043] The antibodies of the invention may be provided in the form ofcompositions for use in passive immunization. While such compositionsare generally administered by injection, it is not intended that thepresent invention be limited to this route alone. In general, however,the compositions of the invention are administered by intramuscular orsubcutaneous injection. Occasionally, the intravenous or intraperitonealroutes may also be used to administer the compositions of the invention.

[0044] In addition to the active ingredient (i.e. the antibody), thecompositions of the invention may also comprise a buffering agent, anagent which adjusts the osmolarity thereof, and optionally, one or morefurther additives, such as carriers, as known in the art.

[0045] A preferred buffering agent is phosphate-buffered saline solution(PBS), which solution is also adjusted for osmolarity.

[0046] A preferred composition is one lacking a carrier. Suchformulations are preferably used for administration by injection,including intramuscular and intravenous injection.

[0047] The preparation of pharmaceutical and immunizing compositions iswell known in the art and has been described in many articles andtextbooks, see e.g., Remington's Pharmaceutical Sciences, Gennaro A. R.ed., Mack Publishing Company, Easton, Pa., 1990.

[0048] It has been shown that the development of autoimmune diabetes inthe NOD mouse is marked by the presence of T-cells reactive to the p277peptide of the HSP 60. It has further been shown that the p277 peptidecan be used as a therapeutic vaccine to arrest NOD diabetes [28]. Thep277 peptide has been shown to arrest also autoimmune diabetes inducedby the Streptozotocin toxin [29]. Likewise, the vaccines according tothe invention may also be used to suppress an autoimmune disease.

[0049] Furthermore, the vaccines of the invention may also be used toprevent relapses of autoimmune diseases, which characterize manyautoimmune diseases. Prevention of a relapse is therefore part of thetherapeutic approach to these disorders. The above peptide p277 has beenshown to prevent NOD mice diabetes by turning off the anti-p277 immunityearly in life. It was later shown to arrest autoimmune process evenafter it is far advanced [28].

[0050] Another possibility is that antibodies against the HSP moleculesuppress inflammation by inhibiting the proinflammatory effect of theHSP on the innate immune system. Mycobacterial HSP65 has been shown toinduce release of pro-inflammatory cytokines from human monocytic cells[18] and the mammalian HSP60 has been shown to synergize with IFN-γ andto promote pro-inflammatory cytokines like IL-12 and IL-15 [31].Induction of anti-Mycobacterial/anti-self HSP antibodies may suppressthose proinflammatory effects.

[0051] Specific immunoglobulins (antibodies) are commonly used forprevention and treatment of infectious diseases (i.e. viral hepatitis).This is termed passive vaccination. Immunoglobulins can also be used tosuppress or prevent relapses of autoimmune diseases like ITP (ImmuneThrombocytopenic Purpura), Myasthenia Gravis (MG), and other autoimmunediseases [30].

[0052] Thus, in yet a further aspect, the invention relates to anantibody directed against at least one peptide according to theinvention or functional homologues and derivatives thereof, which caninduce the production of said antibody.

[0053] The antibodies of the invention may be polyclonal or monoclonalantibodies.

[0054] In yet a further aspect, the invention relates to a compositionfor the passive immunization comprising at least one antibody accordingto the invention, and may optionally further comprise pharmaceuticallyacceptable carriers, diluents, additives, excipients and adjuvants. Thecomposition of the present invention is particularly intended for thepassive vaccination or immunization against, and treatment of autoimmuneor inflammatory disorders, for example, rheumatoid arthritis.

[0055] The invention will be described in more detail on basis of thefollowing Examples, which are illustrative only and do not in any waylimit the invention. Many modifications and variations of the presentinvention are possible in light of the present teachings. It istherefore understood, that within the scope of the appended claims, theinvention may be practiced otherwise than specifically described.

[0056] The following Examples show the anti-MT HSP antibody response ofvarious rats and its correlation with susceptibility to induction ofarthritis. Only a limited number of epitopes in the bacterial HSPmolecule is recognized by rat antibodies. The repertoire of thisantibody differs between resistant and susceptible strains. Resistantstrains were found to respond to peptides that are found on the outersurface of the molecule, as well as to the whole molecule. On the otherhand, antibodies from naive Lewis rats reacted with a smaller number ofpeptides, which are less exposed on the outer surface of the moleculeand did not react with the intact HSP. The presence of antibodiesagainst some of the epitopes, as well as the whole MT-HSP, may beassociated with resistance to the induction of arthritis and they weretherefore named “protective” epitopes.

[0057] It has been previously reported that the T cell response tobacterial HSP shows determinant spreading. The present data, given inthe following Examples, show that there is a clear B cell determinantspreading as well, and this spreading can occur also spontaneously,namely without intentional vaccination. The B cell epitopes, as will beshown, are different from the T cell epitopes. This observation is ofparticular significance to the present invention.

[0058] Young naive Lewis rats recognized only two bacterial epitopes;peptides 40 and 63. Four months Lewis rats recognized, in addition,peptides 6, 36 and 45 and nine months Lewis rats recognized peptides 7and 31, in addition to all the other mentioned peptides. Recognition ofthese peptides is also associated with recognition of the wholebacterial HSP molecule.

[0059] The B cell epitope repertoire of the young BN rats is similar tothat of the old Lewis rats including only one additional peptide,peptide 59. Lewis rats that were immunized with the CFA responded to allthe aforementioned peptides, as well as to two additional peptides,namely 21 and 84.

[0060] Although all the anti HSP peptide antibodies found in naive oldLewis rats and in naive young BN rats are referred to as naturalantibodies, it is possible that they are elicited as a response to theexposure of these rats to environmental pathogens (as “natural”antibodies may indeed always be) and that the epitope spreading inresponse to these pathogens occurs in the BN rat more rapidly, earlierand in more strongly than in the Lewis rat. Lewis rats have to beimmunized with CFA in order to mimic the natural response of the BNrats. The similarity of the antibody repertoire of the naive BN rats tothat of the immunized Lewis rat supports this possibility.

[0061] The nature of the B cell epitopes and the correlation betweenrecognition of certain epitopes and the whole molecule can be betterunderstood from primary and tertiary structure analysis of the molecule,shown hereafter.

[0062] To see whether the anti-HSP protective antibodies can be inducedby immunization with the “protective” peptides, Lewis rats wereimmunized with the various peptides, without Freund's adjuvant.Immunization with three peptides, the bacterial peptides 6 and 7, andthe mammalian peptide 5, led to production of antibodies againstbacterial peptide 6, as well as to an anti-HSP response, showing thatantibodies against an “external” peptide will lead to recognition of thewhole molecule. Induction of these antibodies also led to diseaseresistance.

[0063] Although the mechanism of disease resistance induced by thenatural as well as the induced anti-HSP antibodies has not been yetclarified, it is possible that the antibodies against the MT HSP inhibitthe early steps of induction of pathogenic T cells to the peptide byintervening in the antigen processing or the T cell recognition of thepathogenic epitopes. Alternatively they may prevent the effector stepsof the pathogenic response by binding to the self HSP-cross reactingtarget antigen.

[0064] The T cell response of AA susceptible Lewis and AA resistant WKAWistar rats to the bacterial HSP 65 kD has been thoroughly studied. Ithas been shown that in the early post immunization stages the Lewis Tcells respond to several determinants found in the N terminal, as wellas in the carboxy terminal of the molecule, whereas later a shift tocarboxy terminal epitopes has developed. The early T cell response ofWistar rats was similar to that of the late response of the Lewis rats.As the 3D structure of the molecule does not show the carboxy and the Nterminal sites to be in different locations of the molecule , it is notsurprising that the B-cell epitopes were found all along the moleculewithout any selection of either the carboxy or the N terminal of themolecule.

[0065] A comparison between the published dominant T cell epitopes andthe present B cell epitopes did not reveal common epitopes. To thecontrary, the lack of natural antibodies to certain epitopes like 6, 7or 31 in the naive Lewis rat is associated with an early T cell responseto these epitopes, whereas the presence of antibodies to epitopes like40 and 63 is associated with lack of an early T cell response. Based onthese correlations, it may be suggested that the presence of naturalantibodies to certain epitopes may actually inhibit T cell response tothem, whereas the lack of antibodies enables the T cells to respond tothese epitopes. For example, AA susceptible Lewis rats that do not havenatural antibodies to the bacterial peptide 31 can develop a T cellresponse to this peptide, and these pathogenic T cells can inducearthritis.

[0066] As previously mentioned, there was a clear correlation betweendisease resistance and the presence of anti-HSP antibodies. Young naiveLewis rats did not have detectable antibodies against the HSP moleculewhereas nine months old Lewis rats developed these antibodies in asignificant titer. Parallel to the development of the anti-HSP response,the old rats also became resistant to induction of arthritis. YoungLewis rats acquired both the antibodies and disease resistance afterimmunization with CFA and the naturally resistant BN rats had anti HSPantibodies spontaneously, without the need for immunization. It ispossible therefore that these antibodies bind the bacterial HSPimmediately after immunization and prevent it from becoming accessibleto the cellular arm of the immune system.

[0067] As noted previously, the epitopes “chosen” by the B cells fromthe bacterial HSP are epitopes that have relatively little homology withthe self HSP, most probably as a result of tolerance to self antigens.

[0068] Analysis of the anti self (rat) HSP antibody repertoire indeedshowed that there is a limited number of epitopes recognized by the ratimmunoglobulins in the self HSP molecule. Naive young Lewis rats did notrespond to any self peptide neither did they respond to the whole selfHSP 60 molecule. BN and post-AA Lewis rats that reacted with 8-10bacterial HSP epitopes responded to only two epitopes in the self HSP,peptides M5 and M30, as well as to the whole self HSP molecule.

[0069] Expression of the mammalian (or self) HSP is upregulated ininflamed synovia of rats with AA [22] and cross-reactiveimmunerecognition has been found between the Mycobacterial HSP 65 kD andendogenous self HSP 60 kD at the T-cell level [23-25].

[0070] As the anti self antibodies were found only in the resistantrats, it is possible that antibodies that cross react with the self HSPmay conceal it from the pathogenic T cells and thus act as protectiveantibodies.

[0071] It is interesting to note that one of the two self protectiveepitopes is the self peptide 5, which is the homologous rat epitope tothe bacterial protective peptide 6. Moreover, immunization with thebacterial peptides 6 and 7 and with the mammalian peptide 5 led to theproduction of anti bacterial HSP 6 and anti bacterial HSP antibodies, aswell as protection against disease induction. Observing the primarystructure of these three peptides leads to the conclusion that theyexpress a common motif (V—E—W G—P) which might be the protective motifof these peptides (FIG. 5).

[0072] Therefore, the humoral immune response to the bacterial HSP maybe aimed at a limited number of potential B-cell epitopes. Theseepitopes are peptide stretches located between amino acids that serve asbends and spacers, and are found in non-conserved parts of the molecule.Recognition of B-cell epitopes that are exposed on the surface of themolecule leads to binding to the whole molecule and is associated withresistance to induction of arthritis.

[0073] This resistance occurs naturally in some strains of rats whereasin others it can be acquired with age or upon immunization with HSP.Immunization with some of the “protecting” epitopes can lead both todisease resistance as well as to the serological profile that is presentin the resistant strains.

[0074] The present invention can also provide a method for theprediction of susceptibility/predisposition to develop autoimmunearthritis. In the rat system, it has been shown that naive young Lewisrats do not have antibodies against peptide 6 of the HSP, and that theyare susceptible to the development of arthritis after exposure to orimmunization by HSP. In a similar manner, healthy individuals that lacksub-groups of antibodies against HSP specific peptides may besusceptible to onset of arthritis. The present invention also providesan assay for the assessment and determination ofsusceptibility/predisposition to arthritis. The assay can be performedby ELISA, in which the peptides will be bound to the solid phase andserum samples added, followed by adding anti human Igs. Other knownimmunological analysis techniques can also be used.

[0075] The invention will be described in more detail on hand of thefollowing examples, which are illustrative only and do not limit theinvention thereto.

EXAMPLES Materials

[0076] Animals: Female inbred Lewis rats, 6 weeks or 9 months old, wereobtained from Harlan Lab. Israel. Female Brown-Norway (BN) rats, 6 weeksold, were obtained from Harlan Sprague-Dawley, USA.

[0077] Antigens and antibodies: Recombinant HS P65 of MycobacteriumTuberculosis was a gift from Dr. M. Singh (The WHO Recombinant ProteinBank, Germany). Recombinant mammalian HSP 60 was purchased fromStressGen Biothec. Corp. (Victoria, BC, Canada). Synthetic peptides ofMT HSP 65 were a gift from Dr. L. Adorini (The Roche Milano Ricerche,Milano, Italy). Synthetic peptides of the mammalian HSP 60 were a giftfrom Dr. I. Cohen (The Weizmann Institute, Rehovot, Israel). Goat antiRat IgG conjugated to alkaline-phosphatase was purchased from JacksonImmunoResearch Lab. Inc. (Avonsdale, Pa.).

Methods

[0078] Induction and Clinical Assessment of Adjuvant Arthritis: Lewisrats were injected with 1 mg of Mycobacterium Tuberculosis H37Ra (Difco,Detroit, Mich.) in Complete Freund's Adjuvant (Difco) subcutaneously atthe base of the tail. Severity of Arthritis (arthritis index) wasassessed blindly as follows: 0-no arthritis; 1-redness of the joint;2-redness and swelling of the joint. The ankle and tarsal-metatarsaljoints of each paw were scored. A maximum score of 16 can be obtained,but a score above 8 indicates a severe disease.

[0079] Dot Blots assay: Antigens were dissolved in PBS and samples of 1μg were adsorbed on Nitrocellulose paper. The paper was air-dried andincubated with BSA 1% in PBS for 20 min. to block non-specific binding.The samples were then washed in PBS-Tween 0.05% and incubated with ratsera diluted 1:100 in BSA-PBS, for 90 min. at room temp. Samples werewashed and incubated with goat anti rat antibody conjugated to alkalinephosphatase diluted 1:1000 in BSA-PBS for 90 min. at RT. Afterre-washing the color reaction was developed by adding a mixture ofBCIP-NBT (Sigma-Fast, Sigma) to the cells for 15 min. The reaction wasstopped by the addition of tap water.

[0080] ELISA: Flat-bottomed 96 well plates (Coming) were coated withmammalian HSP 60, or Mycobacterial HSP 65 (10 μg/ml) in carbonate bufferpH 9.6 overnight at 4° C.

[0081] After extensive washing with PBS-Tween 0.05% plates wereincubated with blocking buffer containing 1% BSA (Sigma) for 60 min. atRT.

[0082] HSP peptides were attached to plates pre-treated withglutaraldehyde according to Kasprzyk et al. [26]. Shortly, plates werecoated with 100 μl well of 5% w/v of glutaraldehyde in PBS for 1 hour atroom temp. Plates were washed thoroughly with PBS and peptides (1 μg/100μl) were added to each well, incubated overnight at 4° C. Plates wereshaken dry and blocked with BSA 1% in PBS.

[0083] Plates coated with either HSP or peptides were washed again andincubated with rat sera diluted 1:100 with PBS-Tween 0.01% for 90 min.at room temp. After re-washing the plates were incubated with Goat antirat IgG or IgM conjugated to alkaline-phosphatase for 60 min. at roomtemp. The presence of antibodies was revealed by addition of thesubstrate PNP (NP 100, Chemicon, Temecula, Calif.) to the plates.Optical density was measured photometrically at 405 nm.

[0084] Amino Acid Comparison: “Pileup” and “pretty” programs(GCG—Wisconsin package, v.9.0) were used to compare amino acid sequencesof three HSP 60 (Mycobacterium tuberculosis, rat and human).

[0085] Structure Analysis: RasMol v.2.6 program and the 3D structure ofthe E. Coli complex GroEL-GroES (pdb ID: 1AON reference) were used toanalyze the position of epitopes.

[0086] Since the crystal structure of MT HSP 65 kD is not yet completelyknown, a three-dimensional model for the tertiary structure of MT HSP 65kD based on the solved crystal structure of GroEL from E. Coli (pdb ID:1GRL) was used as template. This model was built by programs forcomparative protein modeling.

[0087] Modulation of AA by mycobacterial and mammalian HSP peptides: HSP65 derived peptides were tested for their ability to modulate theappearance or severity of AA in Lewis rats. Rats were immunized with 100μg of each peptide in PBS, three weeks (3W), 2W and 1W before inductionof AA by MT. Control rats received PBS. Rats were bled for testingantibody presence before injection of MT and 30 days post MT injection.

[0088] DNA Vaccine preparation: A synthetic oligoDNA, having the SEQ IDNO: 5, encoding the oligopeptide Mycobacterium Tuberculosis HSP 65 kDNo. 6, presented in Table 1, was cloned into the commercially availablemammalian expression vector, pTARGET (Programa, Madison, Wis., USA),having the restriction map depicted in FIG. 6. The cloning was carriedout according to the manufacturer's instructions.

[0089] The plasmid construct was then transferred into E. Coli JM109strain and expanded to large scale for further plasmid purification,using the DNA purification system Wizard Plus Maxipreps kit (Programa,Madison, Wis., USA).

[0090] Animal vaccination: Lewis rats were pre-treated with Bupivaccine(Astra) two days prior to vaccination and later disease induction. Therats were then twice injected with 100 μg of the DNA construct, into thetibialis anterior muscle, with a week interval between the injections.

RESULTS The Interaction of Rat Ig with Whole Mycobacterial HSP 65 andits Peptides

[0091] Previous experiments conducted by the inventors showed that Ig'sfrom AA resistant naive rats (i.e. BN or Fisher) as well as Lewis ratsthat recovered from AA (post AA Lewis rats), were able to suppress theinduction of AA in naive Lewis rats and bound to the bacterial HSP 65 ina dot blot assay. To obtain a more quantitative evaluation of thisbinding, the interaction of Ig's from these rats with the whole moleculeof the Mycobacterial HSP 65, known to be associated with AA in Lewisrats, was tested by Dot-Blot and ELISA.

[0092] It was found that Ig's from 6-8 week old BN rats, and post AALewis rats, reacted strongly with the HSP while no reaction was foundwhen Ig's from naive Lewis rats were tested. Interestingly, it was foundthat Ig's from nine months old naive Lewis rat also reacted with theHSP.

[0093] To define the epitopes recognized by the anti bacterial HSPantibodies, the inventors tested by Dot-Blot the interaction of Ig'sfrom naive young BN rats and post AA Lewis rats with 90 16-mer syntheticpeptides of the Mycobacterial 65 kD HSP. Ig's from naive young Lewisrats served as control.

[0094] Only 10 peptides out of the 90 peptides tested (Table 2) reactedwith the immunoglobulins tested. All of the rats immunoglobulins reactedwith two peptides: 40 (residues 235-250) and 63 (residues 373-388). Whenthese rats age, they acquire antibodies against additional peptides, anda similar profile to that of old Lewis rats is found in young naive BNrats, and Lewis rats that were immunized with CFA reacted also withpeptides 21 (residues 121-136) and 84 (residues 499-514). It is notedthat although naive Lewis rats do not recognize the whole molecule ofHSP 65 kD, its Ig's can interact with certain peptides of this molecule,without any effect on susceptibility to AA. TABLE 2 Antibodies toMycobacterial HSP 65 Peptides Peptide Sequence Disease 21 84 59 7 31 636 45 40 63 Suscep- Strain 121-136 499-514 349-364 37-52 181-196 31-46211-226 265-280 236-251 373-388 HSP 65 tibility Lew-6w − − − − − − −− + + − 8/10 Lew-4m − − − − − + + + + + − 3/5  Lew-9m − − − + ++ + + +++ +++ + 0/7  BN-6w − − + + + + ++ + ++ + +++ 0/10 Lew-Post AA + + +++ +++ +++ +++ ++ ++ ++ ++ 0/10

Binding of Rat Ig's with the Mammalian HSP 60 and its Peptides

[0095] Previous studies have shown that certain bacterial HSP peptidesmay trigger self HSP reactive T-cells with disease suppressiveregulatory potential. To analyze the anti self-HSP antibody repertoireof these rats, the reactivity of Ig's from naive and post AA Lewis ratsas well as from naive BN rats to whole mammalian HSP 60 was tested byELISA.

[0096] The results presented in Table 3 indicate that that naive andfour months old Lewis rats do not possess anti self-HSP 60 antibodies,whereas nine months old Lewis rats, young BN rats and post-AA Lewis ratshad significant binding to the self-HSP (Table 3). Some naive Lewis ratshad very low concentrations of the antibodies. TABLE 3 Antibodies toMammalian HSP 60 Peptides Disease Peptide Suscepti- Sequence M 5 61-80 M30 436-455 M-HSP 60 bility Strain Lew-6w − − − 8/10 Lew-4m − − − 3/3 Lew-9m ++ ++ + 0/7  BN-6w + + + 0/10 Lew-Post AA +++ ++ + 0/10

[0097] Immunoglobulins from naive Lewis and BN rats and post-AA Lewisrats were tested for binding to 38 synthetic 20-mer peptides of themammalian HSP 60 by Dot-Blot. It was found that Ig's derived from BN andpost-AA Lewis rats, but not from naive Lewis rats, reacted with 2peptides only: peptide 5 (residues 61-80) and peptide 30 (residues436-455). Quantitative analysis of this binding as well as the bindingof immunoglobulins from four and nine months old Lewis rats confirmedthe dot blot findings (Table 3).

Amino Acid Comparison

[0098] The HSP 60 family is highly conserved: MT-HSP 65 and itsmammalian homologues (rat or human) show 48% identity. In FIG. 1, thethree amino acid sequences of the MT-HSP 65, HSP 60 from rat and humanare compared. The consensus sequence of these three proteins is showntoo. The epitopes that were found to be relevant in this study are shownin Bold and Underlined.

3D Structure Analysis

[0099] Tertiary structure plays an important role for B-cell epitoperecognition. In a first approach, a simple computer program wasprovided, that could predict where to find potential B-cell epitopes byscreening the primary structure of the peptide. The algorithm is basedon a previous analysis by Warren et al. [27] of the Myelin Basic Proteinto locate potential epitopes for B-cell. According to their analysis,two sorts of amino acids can be defined:

[0100] “Molecular spacers”: These are short-chain residues (side chainsof one carbon or less) that could provide a molecular gap for adjacentlong-chain amino acids. Three amino acids that fit this definition are:Glycine (G), Alanine (A) and Serine (S).

[0101] “Molecular bends”: Proline (P) residues that can causedisruptions in secondary structure.

[0102] A minimal length of 9 residues for these potential epitopes wasset. Following these rules, six series of consecutive long-chainresidues (side chains of two carbons or more) located between molecularspacers and/or molecular bends were found (Table 4). TABLE 4 Potentialepitopes of MT HSP 65kD Location of the peptide Experimental (aaresidues) Sequence of the peptide Length peptide matching 35-43G-RNVVLEKKW-G 9 6,7 123-132 A-VEKVTETLLK-G 10 21 135-143 A-KEVETKEQI-A 921 319-332 RKVVVTKDAETTIVE 14 none 357-367 S-DYDREKLQERL-A 11 59 383-396A-TEVELKERKHRIED-A 14 63 183-195 G-LQLELTEGMRFDK-G 13 31 259-270S-TLVVNKIR G TFK-S 12 45

[0103] The peptide was screened by a computer program and consecutivelong-chain residues (side chains of two carbons or more) located betweenmolecular spacers and/or molecular bends are shown (the 6 firstpeptides). The two peptides below them are the consecutive chain thatcontain at most one molecular spacer (glycine).

[0104] Five of six series that were identified by these rules fit aminoacid sequences that were found to be experimentally recognized by B-cellantibodies (Table 1) Consequently, in order to find more epitopes, theprogram was run with a slight change, namely search of epitopes thatcontain at most one molecular spacer (G, S or A). The minimal length wasset at 12 residues (instead of 9 previously) in order to lower thebackground (i.e., a penality of three residues was set to compensate thegap). Two new sequences were identified, that were also found to beexperimentally recognized by B-cell antibodies (31, 45; see Table 1).The molecular spacer was glycine in these two cases.

[0105] In order to better understand the implications of the tertiarystructure of MT HSP 65 kD and to locate these different amino acidsequences on the whole molecule, a model for the tertiary structure ofMT HSP 65 kD based on the crystal structure of E. Coli GroEL (FIG. 3)was used.

[0106] Structure analysis confirmed that the experimentally recognizedepitopes located on the surface of the protein can provide a potentialsite to antibodies binding. Peptides 6, 7, 21, 31, 59 were those thatwere found to be the most exposed whereas peptides 36, 40, 45, 63 and 84are partially exposed.

[0107] The single potential epitope that was not recognizedexperimentally (residues 318-331) seems to be “buried” in the molecule.

[0108] Although there is a marked homology between MT HSP 65 kD andmammalian HSP 60 kD, most of the peptides that were found to berecognized by the anti-MT HSP 65 antibodies did not show high residueshomology with the mammalian HSP. This may be due to the tolerance toself, that protects the rats from developing an autoimmune autoantibodyresponse to their own HSP 60. Two peptides, 6 and 45, did not seem toconform to this rule as they had sites showing high homology to the selfHSP.

[0109] These findings may be explained for both peptides as follows:

[0110] As to peptide 6 (residues 31-46): antibodies were found to bindpeptide 7 (residues 37-52) which overlap the polymorphic part of thispeptide, but not peptide 5 (residues 25-40) representing the regionhomologous with the mammalian HSP. It seems therefore, that theseantibodies are directed against the polymorphic (non-self) region ofpeptide 6 (residues 40-46). It can also provide a hypothesis concerningthe “protective” ability of this peptide, partial homology to themammalian HSP 60 sequence may be responsible for this protective effect.

[0111] As to peptide 45 (residues 265-280): This peptide can be dividedinto two consecutive regions: one polymorphic (residues 265-271) and thesecond highly conserved (residues 271-280). Analysis of thethree-dimensional structure shows that the polymorphic region is theexposed region, whereas the conserved region seems to be “buried” in thewhole molecule (not shown). Therefore, it is possible that theantibodies that bind peptide 45 are mainly directed against the exposedpolymorphic region.

[0112] No particularity concerning the secondary structure and therepartition of hydrophobic/polar residues in these epitopes was noticed(both experimentally and computer recognized). Generally, theexperimentally recognized epitopes tend to be hydrophobic (9-12hydrophobic residues out of 16), but for peptide 59 that is highly polar(13 residues out of 16).

[0113] With reference to the Figures, FIG. 2 shows the location ofbacterial peptides 6, 7 and 31 on the three dimensional structure of theE. Coli GroEL-GroES complex and FIG. 3, as stated, shows the samepeptides on a model of the MT HSP 65 based on the structure of GroEL E.Coli with a space-filling and secondary structure representations.

Analysis of the Ability of Peptides to Immunize Against AA

[0114] To test whether active immunization with bacterial or mammalianHSP peptides that are recognized by protective immunoglobulins caninduce protection against AA, Lewis rats were immunized with themycobacterial peptides 6, 7, 21, 31, 36, 45, 84, that bound antibodiesfrom resistant Lewis rats (“protective” peptides), with somenon-reactive mycobacterial HSP 65 peptides: peptide 26 (residues151-166), 28 (residues 163-178) or peptide 70 (residues 415-430), andwith the mammalian peptide 5.

[0115] Rats were injected 3 times intraperitoneally (IP), with one weekintervals between injections before induction of AA with MT.

[0116]FIG. 4 shows that only pre-immunization of rats with the bacterialpeptides 6 and 7 and the mammalian peptide 5 resulted in a significantsuppression of disease severity.

[0117] Immunization with these “protective” peptides also resulted inthe production of antibodies against peptide 6 as well as against thewhole MT HSP 65 (Table 5). TABLE 5 Anti HSP Antibodies in ImmunizedLewis Rats Immunizing Antigen Peptide 6 7 M5 MT-HSP 65 PBS − − − − 6 ++− − ++ 7 + − − + M5 + − − +

[0118]

1 9 1 22 PRT Mycobacterium tuberculosis 1 Gly Pro Lys Gly Arg Asn ValVal Leu Glu Lys Lys Trp Gly Ala Pro 1 5 10 15 Thr Ile Thr Asn Asp Gly 202 16 PRT Mycobacterium tuberculosis 2 Gly Pro Lys Gly Arg Asn Val ValLeu Glu Lys Lys Trp Gly Ala Pro 1 5 10 15 3 16 PRT Mycobacteriumtuberculosis 3 Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile Thr AsnAsp Gly 1 5 10 15 4 20 PRT Homo sapiens 4 Thr Val Ile Ile Glu Gln SerTrp Gly Ser Pro Lys Val Thr Lys Asp 1 5 10 15 Gly Val Thr Val 20 5 67DNA Homo sapiens 5 gccgccatgg gaccaaaggg acgcaacgtg gtactagagaagaaatgggg cgcgccgtag 60 ctcgaga 67 6 540 PRT Mycobacterium tuberculosis6 Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu 1 5 1015 Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro 20 2530 Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile 35 4045 Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro 50 5560 Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr 65 7075 80 Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln 8590 95 Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro100 105 110 Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val ThrGlu 115 120 125 Thr Leu Leu Lys Gly Ala Lys Glu Val Glu Thr Lys Glu GlnIle Ala 130 135 140 Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile GlyAsp Leu Ile 145 150 155 160 Ala Glu Ala Met Asp Lys Val Gly Asn Glu GlyVal Ile Thr Val Glu 165 170 175 Glu Ser Asn Thr Phe Gly Leu Gln Leu GluLeu Thr Glu Gly Met Arg 180 185 190 Phe Asp Lys Gly Tyr Ile Ser Gly TyrPhe Val Thr Asp Pro Glu Arg 195 200 205 Gln Glu Ala Val Leu Glu Asp ProTyr Ile Leu Leu Val Ser Ser Lys 210 215 220 Val Ser Thr Val Lys Asp LeuLeu Pro Leu Leu Glu Lys Val Ile Gly 225 230 235 240 Ala Gly Lys Pro LeuLeu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala 245 250 255 Leu Ser Thr LeuVal Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val 260 265 270 Ala Val LysAla Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln 275 280 285 Asp MetAla Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly 290 295 300 LeuThr Leu Glu Asn Ala Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys 305 310 315320 Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp 325330 335 Thr Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu340 345 350 Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg LeuAla 355 360 365 Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala AlaThr Glu 370 375 380 Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp AlaVal Arg Asn 385 390 395 400 Ala Lys Ala Ala Val Glu Glu Gly Ile Val AlaGly Gly Gly Val Thr 405 410 415 Leu Leu Gln Ala Ala Pro Thr Leu Asp GluLeu Lys Leu Glu Gly Asp 420 425 430 Glu Ala Thr Gly Ala Asn Ile Val LysVal Ala Leu Glu Ala Pro Leu 435 440 445 Lys Gln Ile Ala Phe Asn Ser GlyLeu Glu Pro Gly Val Val Ala Glu 450 455 460 Lys Val Arg Asn Leu Pro AlaGly His Gly Leu Asn Ala Gln Thr Gly 465 470 475 480 Val Tyr Glu Asp LeuLeu Ala Ala Gly Val Ala Asp Pro Val Lys Val 485 490 495 Thr Arg Ser AlaLeu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu 500 505 510 Thr Thr GluAla Val Val Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser 515 520 525 Val ProGly Gly Gly Asp Met Gly Gly Met Asp Phe 530 535 540 7 573 PRT Rattusnorvegicus 7 Met Leu Arg Leu Pro Thr Val Leu Arg Gln Met Arg Pro Val SerArg 1 5 10 15 Ala Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp ValLys Phe 20 25 30 Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp LeuLeu Ala 35 40 45 Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr ValIle Ile 50 55 60 Glu Gln Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly ValThr Val 65 70 75 80 Ala Lys Ser Ile Asp Leu Lys Asp Lys Tyr Lys Asn IleGly Ala Lys 85 90 95 Leu Val Gln Asp Val Ala Asn Asn Thr Asn Glu Glu AlaGly Asp Gly 100 105 110 Thr Thr Thr Ala Thr Val Leu Ala Arg Ser Ile AlaLys Glu Gly Phe 115 120 125 Glu Lys Ile Ser Lys Gly Ala Asn Pro Val GluIle Arg Arg Gly Val 130 135 140 Met Leu Ala Val Asp Ala Val Ile Ala GluLeu Lys Lys Gln Ser Lys 145 150 155 160 Pro Val Thr Thr Pro Glu Glu IleAla Gln Val Ala Thr Ile Ser Ala 165 170 175 Asn Gly Asp Lys Asp Ile GlyAsn Ile Ile Ser Asp Ala Met Lys Lys 180 185 190 Val Gly Arg Lys Gly ValIle Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205 Asp Glu Leu Glu IleIle Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile 210 215 220 Ser Pro Tyr PheIle Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln 225 230 235 240 Asp AlaTyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Val Gln Ser 245 250 255 IleVal Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275 280285 Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro Gly 290295 300 Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala Ile Ala Thr305 310 315 320 Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Asn Leu Asn LeuGlu Asp 325 330 335 Val Gln Ala His Asp Leu Gly Lys Val Gly Glu Val IleVal Thr Lys 340 345 350 Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp LysAla His Ile Glu 355 360 365 Lys Arg Ile Gln Glu Ile Thr Glu Gln Leu AspIle Thr Thr Ser Glu 370 375 380 Tyr Glu Lys Glu Lys Leu Asn Glu Arg LeuAla Lys Leu Ser Asp Gly 385 390 395 400 Val Ala Val Leu Lys Val Gly GlyThr Ser Asp Val Glu Val Asn Glu 405 410 415 Lys Lys Asp Arg Val Thr AspAla Leu Asn Ala Thr Arg Ala Ala Val 420 425 430 Glu Glu Gly Ile Val LeuGly Gly Gly Cys Ala Leu Leu Arg Cys Ile 435 440 445 Pro Ala Leu Asp SerLeu Lys Pro Ala Asn Glu Asp Gln Lys Ile Gly 450 455 460 Ile Glu Ile IleLys Arg Ala Leu Lys Ile Pro Ala Met Thr Ile Ala 465 470 475 480 Lys AsnAla Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Leu Gln 485 490 495 SerSer Ser Glu Val Gly Tyr Asp Ala Met Leu Gly Asp Phe Val Asn 500 505 510Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala 515 520525 Leu Leu Asp Ala Ala Gly Val Ala Pro Leu Leu Thr Thr Ala Glu Ala 530535 540 Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala545 550 555 560 Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565570 8 573 PRT Homo sapiens 8 Met Leu Arg Leu Pro Thr Val Phe Arg Gln MetArg Pro Val Ser Arg 1 5 10 15 Val Leu Ala Pro His Leu Thr Arg Ala TyrAla Lys Asp Val Lys Phe 20 25 30 Gly Ala Asp Ala Arg Ala Leu Met Leu GlnGly Val Asp Leu Leu Ala 35 40 45 Asp Ala Val Ala Val Thr Met Gly Pro LysGly Arg Thr Val Ile Ile 50 55 60 Glu Gln Ser Trp Gly Ser Pro Lys Val ThrLys Asp Gly Val Thr Val 65 70 75 80 Ala Lys Ser Ile Asp Leu Lys Asp LysTyr Lys Asn Ile Gly Ala Lys 85 90 95 Leu Val Gln Asp Val Ala Asn Asn ThrAsn Glu Glu Ala Gly Asp Gly 100 105 110 Thr Thr Thr Ala Thr Val Leu AlaArg Ser Ile Ala Lys Glu Gly Phe 115 120 125 Glu Lys Ile Ser Lys Gly AlaAsn Pro Val Glu Ile Arg Arg Gly Val 130 135 140 Met Leu Ala Val Asp AlaVal Ile Ala Glu Leu Lys Lys Gln Ser Lys 145 150 155 160 Pro Val Thr ThrPro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala 165 170 175 Asn Gly AspLys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys 180 185 190 Val GlyArg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205 AspGlu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile 210 215 220Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln 225 230235 240 Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser245 250 255 Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro LeuVal 260 265 270 Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr LeuVal Leu 275 280 285 Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val LysAla Pro Gly 290 295 300 Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp MetAla Ile Ala Thr 305 310 315 320 Gly Gly Ala Val Phe Gly Glu Glu Gly LeuThr Leu Asn Leu Glu Asp 325 330 335 Val Gln Pro His Asp Leu Gly Lys ValGly Glu Val Ile Val Thr Lys 340 345 350 Asp Asp Ala Met Leu Leu Lys GlyLys Gly Asp Lys Ala Gln Ile Glu 355 360 365 Lys Arg Ile Gln Glu Ile IleGlu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380 Tyr Glu Lys Glu Lys LeuAsn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385 390 395 400 Val Ala Val LeuLys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu 405 410 415 Lys Lys AspArg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 425 430 Glu GluGly Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile 435 440 445 ProAla Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gln Lys Ile Gly 450 455 460Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile Pro Ala Met Thr Ile Ala 465 470475 480 Lys Asn Ala Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln485 490 495 Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe ValAsn 500 505 510 Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val ArgThr Ala 515 520 525 Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr ThrAla Glu Ala 530 535 540 Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp ProGly Met Gly Ala 545 550 555 560 Met Gly Gly Met Gly Gly Gly Met Gly GlyGly Met Phe 565 570 9 255 PRT Artificial Sequence Common motif 9 Ala LysAla Arg Gly Leu Ala Asp Ala Val Val Thr Gly Pro Lys Gly 1 5 10 15 ArgVal Glu Trp Gly Pro Thr Asp Gly Val Ala Lys Ile Leu Asp Tyr 20 25 30 IleGly Ala Leu Val Val Ala Thr Ala Gly Asp Gly Thr Thr Thr Ala 35 40 45 ThrVal Leu Ala Glu Gly Gly Ala Asn Pro Arg Gly Ala Val Val Leu 50 55 60 LysLys Val Thr Glu Ile Ala Ala Ile Ser Ala Gly Asp Ile Gly Ile 65 70 75 80Ala Met Lys Val Gly Gly Val Ile Thr Val Thr Leu Glu Glu Gly Met 85 90 95Phe Asp Gly Tyr Ile Ser Tyr Phe Gln Asp Tyr Leu Leu Lys Ser Pro 100 105110 Leu Glu Lys Pro Leu Ile Ile Ala Glu Asp Val Gly Glu Ala Leu Ser 115120 125 Thr Leu Val Asn Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Lys130 135 140 Leu Asp Met Ala Ile Thr Gly Gly Val Glu Glu Leu Leu Glu LeuGly 145 150 155 160 Lys Val Val Thr Lys Asp Gly Gly Asp Ile Arg Ile SerTyr Glu Lys 165 170 175 Leu Glu Arg Leu Ala Lys Leu Gly Val Ala Val LysGly Val Glu Glu 180 185 190 Lys Arg Asp Ala Ala Ala Val Glu Glu Gly IleVal Gly Gly Gly Leu 195 200 205 Leu Pro Leu Asp Leu Asp Gly Ile Lys LeuPro Leu Ala Asn Gly Glu 210 215 220 Glu Lys Gly Ala Gly Gly Asp Pro LysVal Arg Ala Leu Ala Ala Ala 225 230 235 240 Leu Thr Glu Val Val Pro GluLys Pro Gly Gly Met Gly Gly Met 245 250 255

What is claimed is:
 1. A B cell epitope peptide comprising the aminoacid sequence as denoted by SEQ ID NO:1.
 2. A B cell epitope peptide asclaimed in claim 1 having the amino acid sequence as denoted by SEQ IDNO:2.
 3. A B cell epitope peptide as claimed in claim 1 having the aminoacid sequence as denoted by SEQ ID NO:3.
 4. A B cell epitope peptidecomprising the amino acid sequence as denoted by SEQ ID NO:4.
 5. A Bcell epitope peptide as claimed in claim 1, being a synthetic peptide.6. A chemically modified peptide according to claim
 5. 7. A B cellepitope peptide as claimed in claim 1 capable of conferring immunityagainst autoimmune and/or inflammatory disorders.
 8. A nucleic acidsequence encoding a B cell epitope peptide as claimed in claim
 1. 9. ADNA sequence as claimed in to claim
 8. 10. A DNA sequence as claimed inclaim 9 comprising the nucleic acid sequence as denoted by SEQ ID NO:5.11. A DNA construct comprising a nucleic acid sequence as claimed inclaim
 8. 12. A DNA construct comprising the DNA sequence as claimed inclaim
 9. 13. A vaccine comprising as active ingredient an effectivevaccinating amount of at least one B cell epitope peptide as claimed inclaim 1, optionally further comprising pharmaceutically acceptablecarrier, diluent, additive or adjuvant.
 14. A vaccine comprising asactive ingredient a DNA sequence as claimed in claim 1, optionallyfurther comprising a pharmaceutically acceptable carrier, diluent,additive or adjuvant.
 15. A vaccine as claimed in claim 13, forconferring immunity against autoimmune or inflammatory disorders. 16.The vaccine as claimed claim 15 for conferring immunity againstarthritis.
 17. An antibody directed against a B cell epitope peptidecomprising the amino acid sequence as denoted by SEQ ID NO:1.
 18. Anantibody directed against a B cell epitope peptide having the amino acidsequence as denoted by SEQ ID NO:2.
 19. An antibody directed against a Bcell epitope peptide having the amino acid sequence as denoted by SEQ IDNO:3.
 20. An antibody directed against a B cell epitope peptidecomprising the amino acid sequence as denoted by SEQ ID NO:4.
 21. Acomposition for the passive immunization against an autoimmune orinflammatory disorder comprising a pharmaceutically effective amount ofan antibody as claimed in claim
 17. 22. A composition as claimed inclaim 21, for the prevention or treatment of autoimmune or inflammatorydisorder.
 23. A method for predicting the susceptibility/predispositionof an individual to arthritis by testing a sample of serum from saidindividual for the presence of antibodies directed against a peptide asclaimed in claim 1, by suitable immunoassay techniques.